Regulation of nitrate reductase during in vitro differentiation of nicotiana tabacum L. var. samsun.

Abstract

The commencement of in vitro differentiation is mediated by genetic changes that result in selective expression of genes and a shift in metabolism. The role of nitrate reductase, a key enzyme of nitrate assimilation, during differentiation was examined in this study using an in vitro Nicotiana tabacum (tobacco) callus culture system. In particular, the effects of nitrogen and light/dark regimes on callus differentiation and nitrate reductase were investigated. Methodology required for the analysis of nitrate reductase regulation during in vitro tobacco callus differentiation was established. Optimised in vivo, in situ and in vitro nitrate reductase assays yielded similar values and patterns during tobacco callus culture development, and the in vivo assay was selected for nitrate reductase activity measurement during subsequent experiments. Western blot analysis of tobacco callus acetone-extracted protein after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a spinach polyclonal nitrate reductase antibody yielded major bands at 71 and 48 kD, with numerous minor bands. Extraction of callus protein in the presence of various protectants did not prevent cleavage of putative nitrate reductase polypeptide. Slot blot detection of nitrate reductase mRNA using a [32p]- labelled nitrate reductase cDNA probe isolated from the plasmid pBMC102010 was not possible due to non-specific binding to nitrocellulose filters. Northern blotting of RNA fractionated by agarose gel electrophoresis using a [32p]-labelled nitrate reductase cDNA probe identified a single mRNA species at 3.5 kb, the expected size of tobacco nitrate reductase mRNA. In vitro tobacco callus differentiation on 60 or 120 mM nitrogen regimes and under light/dark (16/8 h), continuous dark or continuous light treatments were comparable in terms of fresh weight, protein and nitrate uptake. Higher levels of in vivo nitrate reductase activity were observed prior to visible shoot primordia in all treatments, suggesting that the developmental status of callus mediated the regulation of nitrate reductase. Putative nitrate reductase protein levels were not correlated with in vivo nitrate reductase activity during initial stages of tobacco callus differentiation under various light treatments; nitrate reductase mRNA levels could not be ascertained. These results suggested that post-translational control mechanisms were involved in nitrate reductase regulation during in vitro tobacco callus differentiation.